Method for determining p1/p2 blood type and detection kit thereof

ABSTRACT

The present invention provides a method for determining P 1 /P 2  blood type, including steps of providing a biological sample of a subject, detecting a genotype for single nucleotide polymorphism rs2143918 or rs5751348 in A4GALT gene of the biological sample and determining a phenotype of the subject based on the genotype. Further, the present invention also provides a kit for determining P 1 /P 2  blood type, including a primer pair for detecting a genotype for single nucleotide polymorphisms rs2143918 or rs5751348 in A4GALT gene of a nucleic acid sample of a subject.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for determining P₁/P₂ bloodtype and a detection kit thereof, and more particularly to a method witha single nucleotide polymorphism for determining P₁/P₂ blood type and adetection kit thereof.

2. Description of Related Art

In 1927, Austrian Biologist Karl Landsteiner and American immunologistPhilip Levine discovered a new antigen from the rabbits transfused humanblood during research on hemolytic disease of the newborns. Theseantigens can cause clotting of the red blood cells in some people whileother groups of people are unaffected. They namely these two blood typesas P₁ (+) and P₁ (−), later, the P1 (+)- and P1 (−) type are referred toas P1 and P2 type. This P₁/P₂ blood type belongs to the third systemamong the present 33 human blood type.

P₁/P₂ blood type is a system composed of glycolipid antigens; howeverthe molecular genetic basis is still not clear. P₁ blood type and P₂blood type are correlated to P₁ and P^(k) blood antigens expressed onthe red blood cell. The P₁ and P^(k) are carbohydrate antigens and aresynthesized via different biosynthetic routes, starting from a commonglycosphingolipid precursor, lactosylceramide. The P₁ and P^(k) antigensare determined by the enzymatic activity of α-1,4-galactosyltransferase(hereinafter referred to as A4GALT). P₁/P₂ blood type is a commonphenotypic polymorphism and the distribution of which varies amongdifferent human populations. The frequency of P₁ type in Caucasians andAfricans are 80% and 90-95%, respectively, while the frequency of P₁type is relatively low in Asians. The frequency of P₁ blood type isabout 30% to 40% in Taiwan.

Using DNA genotype as a method for detecting blood type will be a trendin the future. For instance, Progenika Biopharma, S.A. (Spain) hasdeveloped a blood chip and such blood chip contains various genotypevariations of different blood type. However, in the past, the moleculargenetic mechanism of P₁/P₂ blood type has not been verified, such thatthe current DNA test including Progenika blood chip could not containthis common P₁/P₂ blood type polymorphism to perform DNA analysis.Therefore, there still is a need for determining the P₁/P₂ blood type.

Thuresson et al. (Thuresson B, Westman J S, Olsson M L, Identificationof a novel A4GALT exon reveals the genetic basis of the P₁/P₂histo-blood groups. Blood. 2011; 117(2): 678-687) has reported theassociation between SNP rs8138197 and P₁/P₂ phenotypes, and proposed anovel molecular model of the formation of P₁/P₂ blood group.

The present invention utilizes a genetic association study amongdifferent ethnic populations to prove single nucleotide polymorphism(hereinafter referred to as SNP) rs2143918 and rs5751348 in A4GALT geneare completely associated with P₁/P₂ blood type. The result of thepresent invention provides the molecular genetic mechanism of P₁/P₂blood type and further provides a marker for determining P₁/P₂ bloodtype by using the genotypes of SNPs rs2143918 and rs5751348.

SUMMARY OF THE INVENTION

The present invention aims to conduct a pilot investigation among fourethnic groups, and the results from analysis of P₁ type of Africans anda Taiwanese family pedigree labeled M3 reveals that a genotype of SNPrs8138197 is not associated with P₁/P₂ blood type.

The present invention provides a method for determining P₁/P₂ bloodtype, comprising steps of: providing a biological sample of a subject;detecting a genotype for a single nucleotide polymorphism at rs2143918or rs5751348 in A4GALT gene of the biological sample; and determining aphenotype of the subject based on the genotype.

In one embodiment of the present invention, the genotype of the singlenucleotide polymorphism at rs2143918 is selected from the groupconsisting of T/T, T/G and GIG.

In one embodiment of the present invention, T/T or T/G genotype of thesingle nucleotide polymorphism at rs2143918 represents P₁ phenotype inthe subject.

In one embodiment of the present invention, G/G genotype of the singlenucleotide polymorphism at rs2143918 represents P₂ phenotype in thesubject.

In one embodiment of the present invention, the genotype of the singlenucleotide polymorphism at rs5751348 is selected from the groupconsisting of G/G, G/T and T/T.

In one embodiment of the present invention, G/G or G/T genotype of thesingle nucleotide polymorphism at rs5751348 represents P₁ phenotype inthe subject.

In one embodiment of the present invention, T/T genotype of the singlenucleotide polymorphism at rs5751348 represents P₂ phenotype in thesubject.

In one embodiment of the present invention, the biological samples areblood or saliva.

In one embodiment of the present invention, a nucleic acid in thebiological sample is determined by polymerase chain reaction(hereinafter referred to as PCR).

In one embodiment of the present invention, a primer pair of SEQ ID Nos:1 and 2 are used in the PCR for determining the nucleic acids of thebiological samples.

In one embodiment of the present invention, a primer pair of SEQ ID Nos:3 and 4 are used in the PCR for determining the nucleic acids of thebiological samples.

The present invention also provides a detection kit for determiningP₁/P₂ blood type, comprising: a primer pair for determining a genotypeof a single nucleotide polymorphism at rs2143918 or rs5751348 of A4GALTgene in a nucleic acid sample of a subject.

In one embodiment of the present invention, the primer pair hassequences of SEQ ID Nos: 1 and 2.

In one embodiment of the present invention, the primer pair hassequences of SEQ ID Nos: 3 and 4.

In one embodiment of the present invention, the genotype of the singlenucleotide polymorphism at rs2143918 is selected from the groupconsisting of T/T, T/G and GIG.

In one embodiment of the present invention, T/T or T/G genotype of thesingle nucleotide polymorphism at rs2143918 represents P₁ phenotype inthe subject.

In one embodiment of the present invention, G/G genotype of the singlenucleotide polymorphism at rs2143918 represents P₂ phenotype in thesubject.

In one embodiment of the present invention, the genotype of the singlenucleotide polymorphism at rs5751348 is selected from the groupconsisting of G/G, G/T and T/T.

In one embodiment of the present invention, G/G or G/T genotype of thesingle nucleotide polymorphism at rs5751348 represents P₁ phenotype inthe subject. In one embodiment of the present invention, T/T genotype ofthe single nucleotide polymorphism at rs5751348 represents P₂ phenotypein the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows distributions of the most likely haplotype pairs in P₁/P₂individuals across 4 ethnic populations; and

FIG. 2 shows P₁/P₂ phenotypes and the most likely haplotype pairs of M3family.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following specific examples are used for illustrating the presentinvention. A person skilled in the art can easily conceive the otheradvantages and effects of the present invention. The present inventioncan also be implemented by different specific cases be enacted orapplication, the details of the instructions can also be based ondifferent perspectives and applications in various modifications andchanges do not depart from the spirit of the creation.

The present invention provides a method for determining P₁/P₂ bloodtype, comprising steps of providing a biological sample of a subject;determining a genotype for a single nucleotide polymorphism (SNP) ofA4GALT gene from the biological samples, wherein the SNP is located atSNP rs2143918 or SNP rs5751348; and discriminating the subject has P₁ orP₂ phenotype according to the genotype of SNP rs2143918 or SNPrs5751348.

The present invention also provides a kit for determining P₁/P₂ bloodtype, comprising a PCR primer pair, which is used to amplify nucleicacid fragments of a single nucleotide polymorphism of A4GALT gene byusing the nucleic acid of the subject to be determined as a template.The single nucleotide polymorphism is located at SNP rs2143918 or SNPrs5751348, wherein the PCR primer pair can perform an extension reactionof the primer so as to determine the genotype of SNP rs2143918 or SNPrs5751348 and discriminate the subject has P₁ or P₂ phenotype.

Example 1 Method for Determining P₁/P₂ Phenotype

1. Sample Preparation and P₁/P₂ Phenotype

A total of 338 non-related subjects across four ethnic populations,including 227 Taiwanese, 32 Indians, 46 Caucasians, and 33 Africans(Blacks, including 2 African-Americans) were enrolled in the presentinvention. Peripheral blood samples were collected from each subject.The genomic DNAs were purified from the peripheral blood samples usingQIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany), and then theP₁/P₂ phenotype of each sample was determined by a standardhemagglutination test using monoclonal anti-P1 reagent (Immucor Inc.,Houston, Tex.).

2. Analysis of the Single Nucleotide Polymorphism

57 DNA fragments were amplified by polymerase chain reaction(hereinafter referred to as PCR), and these encompassed all SNP sitesdistribute from −7.0 kb to +17.3 kb of the A4GALT gene. The sequences ofPCR primer pairs locations thereof are listed in Table 1. Then, thenucleotides at the SNP sites were determined by direct sequencing theamplified DNA fragments using Sanger's method.

TABLE 1 PCR primers for SNP analysis primer sequence(5′→3′) location^(a)930R GTGCATGCCTGTAATCACAG −7085 930F GTGTCACAGTGACTGCTGTG −6765 217FATTGACAAGGGCGAGCCAC −6800 217R AAGCAAACACTCCTCCCTCC −6618 928RATGGGTGGAGTGGAAAGTG −6564 928F AAGACAGAGTCTCCCTGTC −6236 931FTGTGGCTCTGACTACTGAG −5713 931R ACACACCTAGAAGCCATCC −5983 678FGCAGTGGCTCCCTTGACATAAGTAACTCC −5265 677R TTATGTCAAGGGAGCCACTG −5285 677FGTCAACATCTAGACCACTGC −4950 584F ACCAGAAAGTTGGAGCTAGG −5129 584RAACTGGAGCTGGTCATCTGG −4521 331R ATCTGCTTTGGAGCATGGGC −4834 331FCTGGGATTACAGGCGTGAGC −4437 853R CAGCTCCAGTTTACTGATGG −4532 853FGTCAGGCTGGTCTTGATCAC −4056 690F1 CAAGACCAGCCTGACCAACG −4070 690R2GGTTGAGCACACAGGCTCTG −3480 628R2 CTCTCAGAGCCTGTGTGCTC −3503 628F1AGCCCCAGCAGCCTTTGAG −3193 644F AATGATCCTACTGCCTCAGC −3078 676FTATTCAAGGCAGGCCAGCAC −2182 676R TCGCTCTGTCGCCATACTGG −2684 676FTATTCAAGGCAGGCCAGCAC −2182 795F CTCAGCCTCCCACAGTGTGG −1607 795RGACGGAGTCTCACTCTGTCG −2138 326R GTTGCCTTCTAGTCTCTGAG −1783 326FGAAGAGACTCACTCTGTTACC −1383 035R TTAGCCATGGTTGTGCATGCCTGTAGTCC −1524035F AGGCCAGGCAAGCACTCACGCCTGTAATC −1056 369R ATCTCAGGTGACCTGCTTGC −1125369F GGGTGACTTCTTGAGTGCTG −680 898R ACCCATTGAGTGCCAGGCTC −799 898FACCGTGCTAAGGGCATTGCC −373 A4GTFa CACCAGGACTGTGACATGCTGGAAACATGG −626PKRL CTGCCTCGAACCCGGCTTCT +430 469F3 GGAGAAGCCGGGTTCGAGGCAGGCTCTGC +409469R4 GCTTTAACCCACTCAGGGCCAGAGGCTCAGC +939 845R GTGAGGATTTGGACCTGCCC+596 927F ACTACAGTGAGCTGCCACAG +1472 573R CAACGTCGAGTGATTGTTCC +1002573F GGTTGCAGCTATGGAAACAC +1303 927R ATAGCAGAGTGCGGATGGAG +1188 927FACTACAGTGAGCTGCCACAG +1472 672R TGTAGTCTCAAACTGCTAGG +1467 672FCAATCCTGCTCCACACTGG +1783 920R GCTGCTAGGTTAATGGGTCC +1710 920FTTCAGGAAGCACCTGCTAGG +2186 522R AAGTGCACCTCCTCTCACTC +1985 522FCTCACTGCACTCTCTGCCTC +2538 175F TCTAGCTTTCCCATCAGC +3239 926RGACAGAGAGATAGAGAGGAG +3132 926F TATGTGGACACTGGTCTGG +3800 991RCCTTCCAGACCAGTGTCCAC +3778 991F AACAGAGCATGGTGGCTGAG +4136 181RCCCTGATCATCTGTGACC +4018 181F AAAAGCTCACTGTCAGGC +4501 181FsCTATTAAACCACACAGCTCC +4462 860F ATTAGCAGGGAATGGTGG +5307 183FGAGTGCACTGGTGCAATCATG +5189 183R CTTCATACCATAAATTCCAAG +5674 894RGTCCTTCAGCAGCTCTCAAG +5750 892R TGGCTCCCTCCTGTAATTC +6553 176FGGAGTGCGGTGGTATGAG +6627 858R ATCTGGCAAAACCCCACCTC +7245 245FCCTAACCTCAGGTGATCCACC +7267 245R CTCCCCCAGCATCACCTAC +7915 718FCCCCTTCTCAGGCAGTATCC +7843 718R CACCTTCGCTCTGGACAC +8550 291FTTGCATCTCTGGGATCTCTG +8429 291R AGTACCTGACTACTTGCCAG +8760 888FTGAGAAGCCAGCCCCACC +8637 888R CCGAGTCTCGCTCTGTTGCC +9215 185FGAGGTTGTAGTGAGCCAAG +9153 185R CTGTGAGCAAACAGGCATG +9533 306FTGTGGTAGGATCTGTGCTGG +9272 306R TAGCTACTCAGGAGGCTGAG +9942 887RTGCAGCAAGCCAAGATGGTG +10216 887F AGCCTCCTGAGTAGCTAGGG +9926 081FCATCTTGGCTTGCTGCAGCC +10200 081R ACAGTTTCATACCTGGGCAC +10779 347RGCTCAAGCAATCTGGCTGCC +10368 347F ACACAGAAGCCAGGAACCAG +10825 793FTGGTGGTGGCAGCATCTGTG +10719 793R GGCAGGTGGATCACTTGAGG +11293 653FGATTACAGTTGTGCCCCACC +11179 479R CAAGTACATGATCCTCCCAC +11697 616FTGGGAGCCTAGGAATTCAAG +11563 616R GGATCCCAGAAGACATAGC +11988 993FGTAGATTAGCTATGTCTTCTGGG +11962 993R ATCGCGCCATTGCATTCTAG +12521 227FCTGGCACTGCAGGTACACAC +12422 227R ACACAGAAACATGGCTGGTC +13012 634FATTGTTACATACACTGGTGG +13082 634R ACTCTCTACCCTAGTGATAG +13708 280FGCACAGTATCTATCACTAGG +13680 280R GAGACATACCTTAAACGAAG +14387 712RATCCTGGCTAACACGGTG +14693 036F TCAGCCTCCCGAGTAACTG +14594 036RTCGCTTGAACCTGGGAAGTG +14884 903F CCTCGGCCTATTAAAGTGC +15079 903RTGAGGAGCACAAATACTCGC +15590 557F AGCGAGTATTTGTGCTCCTC +15570 557RAAGACCCCAGAAAAGGCC +15934 881F AAAGGCTCCCTCCTCTGTTG +15731 881RGAAGCCAGGAATCAAGCAGG +16230 436F TCTGTTTGTAACGTCCACCC +16177 436RCCTTCACTGCTTTGTCCATC +16889 193F GCAGGGTTTGGAAGCTCTGG +16787 193RTGTGCCCGGCCTGCAATAAG +17308 ^(a)Transcription initiation nucleotide ofA4GALT exon 1 of RefSeq Transcript

3. Primer Pairs for Amplification and Identification of the Genotypes ofrs2143918 and rs5751348

-   -   The forward primer for amplification and identification the        genotype of rs2143918 has the sequence of SEQ ID No. 1, while        the reverse primer has the sequence of SEQ ID No. 2.

AAGTGCACCTCCTCTCACTC (SEQ ID NO. 1) TCTAGCTTTCCCATCAGC (SEQ ID NO. 2)

-   -   The forward primer for amplification and identification the        genotype of rs5751347 has the sequence of SEQ ID No. 3, while        the reverse primer has the sequence of SEQ ID No. 4.

TCACGAGCATTCCTCATC (SEQ ID NO: 3) CTCCTCTCTATCTCTCTGTC (SEQ ID NO: 4)

4. Result

1) A Pilot Investigation for Identification of SNPs in A4GALT GeneAssociated with the P₁/P₂ Phenotype.

In order to explore the molecular genetic basis of the P₁/P₂ bloodgroups, the present invention conducted a pilot investigation, whichinvolved the detailed and stepwise screening of SNPs in A4GALT gene fromfour Taiwanese with the P₁ phenotype and four Taiwanese with the P₂phenotype. The screening of SNP started from the 5′ promoter region andthen extended stepwise to the 5′ and 3′ region of the gene to identifyany polymorphic nucleotide positions that may be related to P₁/P₂phenotypes. The PCR, which was used to amplify the DNA fragmentsencompassing each SNP in the A4GALT gene, has been recorded in the SNPdatabase of the National Center for Biotechnology Information (NCBI).Eventually, 57 DNA fragments were amplified respectively by PCR. Theamplified DNA fragments contain a total of 416 different SNP sites whichwere distributed over 24.3 kb region of the A4GALT gene including 7.0 kbof the 5′ promoter region, exon 1 (74 bp), and 17.3 kb of the 5′ part ofintron 1. The nucleotides at each SNP over the eight samples weredetermined and the results are shown in Table 2. These show that 11 SNPsits of 8 subjects exhibited an association with the P₁/P₂ phenotype.These 11 SNPs are distributed from the +1.3 kb to +11.5 kb region in theintron 1 of the A4GALT gene, and they are denoted as SNP1 to SNP11 from5′ to 3′.

TABLE 2 SNPs in the A4GALT gene that show an association with the P₁/P₂blood group phenotypes in the pilot investigation SNP1 SNP2 SNP3 SNP4SNPS SNP6 Location^(a) +1352 +2326 +2414 +2837 +2857 +3084 SNPIDrs67162484^(b) rs8138197 rs10713068 rs2143919 rs2143918 rs5751348rs66781836^(b) Nucleotide AGAA/—    C/T  C/— C/G  T/G G/T Polymorphisms^(c) Phenotype Sample Genotype^(c) P₁ M8 +/+ C/C C/C C/CT/T G/G M5 +/+ C/C C/C C/C T/T G/G M33 +/− C/T  C/— C/G  T/G G/T  HSC+/− C/T  C/— C/G  T/G G/T  P₂ M12 −/− T/T —/— G/G G/G T/T T9 −/− T/T —/—G/G G/G T/T J1 −/− T/T —/— G/G G/G T/T Y1 −/− T/T —/— G/G G/G T/T SNP7SNP8 SNP9 SNP10 SNP11 Location^(a) +3443 +5012 +6762 +7038 +11507 SNPIDrs9623671 rs111626860 rs5758891 rs8139674 rs66463955 Nucleotide G/AACTCCA/—    T/C G/T   G/— Polymorphisms^(c) Phenotype SampleGenotype^(c) P₁ M8 G/G +/+ T/T G/G G/G M5 G/G +/+ T/T G/G G/G M33 G/A+/− T/C G/T   G/— HSC G/A +/− T/C G/T   G/— P₂ M12 A/A −/− C/C T/T —/—T9 A/A −/− C/C T/T —/— J1 A/A −/− C/C T/T —/— Y1 A/A −/− C/C T/T —/—^(a)Transcription initiation nucleotide of A4GALT exon 1 (74 bp) ofRefSeq Transcript NM_017436.4 in the NCBI database as +1 ^(b)SNPsrs67162484 and rs66781836 yield identical sequence polymorphisms.^(c)“+” and “−” indicate the presence and the absence of thenucleotide(s), respectively.

2) SNPs rs2143918 and rs5751348 Show a Definite Association with theP₁/P₂ Phenotypes Across Different Ethnic Populations.

In order to verify the association of these 11 SNPs (SNP1 to SNP11) andthe phenotypes of P₁/P₂ phenotypes, a large scale association studyusing various ethnic populations was carried out There were 227Taiwanese (including 8 subjects analyzed in the previous pilot study),32 Indians, 46 Caucasians and 33 Africans (Black) enrolled in thisstudy. The P₁/P₂ phenotypes and the genotypes at SNP1 to SNP11 of these338 subjects were determined. The distributions of the genotypes at the11 SNPs in the P₁ and P₂ individuals of each population are shown inTable 3. The haplotypes of the 11 SNPs in the 4 populations werereconstructed and the most likely haplotype was assigned for eachsubject using PHASE program (version 2.1). FIG. 1 shows the distributionof the most likely haplotype pairs in the P₁ and P₂ subjects among eachethnic population.

The two haplotypes identified by using 8 Taiwanese in the pilot studywere found to be major haplotypes present in the wider study and werealso associated with the P₁ and P₂ phenotypes across all fourpopulations. In addition to the two haplotypes found in the fourpopulations, haplotypes with recombination between the 11 SNPs wereidentified and were found to be more frequent in Caucasians and Africansthan in Taiwanese and Indians. The correlation of SNP1, SNP3, SNP4, andSNP7 to SNP11 could be easily excluded while multiple cases distributedacross the different ethnic populations were identified as recombinanthaplotypes between these SNPs, as shown in Table 3 and FIG. 1. Thus, itcreates inconsistencies in the genotype-phenotype correlation observedacross the majority of other enrolled subjects.

Among the 338 enrolled subjects, only one subject (P₁ Africans, denotedas ** in Table 3 and * in FIG. 1) showed the genotype-phenotypeinconsistency of SNP rs8138197. In the previous analysis for the P₁ andP₂ phenotype from 208 Swedish subjects, there was one case found thegenotype-phenotype inconsistency of SNP rs8138197. In addition to suchP₁ African case, a P₁ Taiwanese (denoted as * in FIG. 1) labeled as M3family was pointed out as having an allele which is involvedrecombination between SNP3 and SNP4. As shown in FIG. 2, the M3 familywas then enrolled to perform the pedigree analysis and the recombinantallele identified in M3 was found to be present in M3's father andbrother who were labeled with the P₂ phenotype. The results obtainedfrom this pedigree analysis suggest that SNP2 (i.e. rs8138197) is norinvolved in defining the P₁ and P₂ phenotypes.

When the above results were taken together, the SNPs that show definiteassociation with the P₁/P₂ phenotypes could be narrowed down to two,namely SNP5 (rs2143918) and SNP6 (rs5751348), using this pilot studyacross different ethnic populations. The genotypes at the SNPsdistributed along the +1.3 kb through +3.7 kb region of the A4GALT gene,which encompasses the SNP1 to SNP7 sites, of the enrolled 338 subjectswere thoroughly examined, and no other SNP was found an association withthe P₁ and P₂ phenotypes. The genotypes at SNPs rs2143918 and rs5751348were consistently associated with the P₁ and P₂ phenotypic polymorphismswhile 338 subjects across the four ethnic populations have considerablegenetic distances.

TABLE 3 SNPs associated with the P₁/P₂ blood group phenotypes anddistributions of the SNP genotypes in the P₁ and P₂ subjects of variousethnic populations SNP1 SNP2 SNP3 SNP4 SNP5 SNP6 Location^(a) +1352+2326 +2414 +2837 +2857 +3084 SNP ID rs67162484^(b) rs8138197 rs10713068rs2143919 rs2143918 rs5751348 rs66781836^(b) Nucleotide AGAA C C C T GPolymorphisms^(c) (▪)/—(□) (▪)/T(□) (▪)/—(□) (▪)/G(□) (▪)/G(□) (▪)/T(□)▪/ ▪/ □/ ▪/ ▪/ □/ ▪/ ▪/ □/ ▪/ ▪/ □/ ▪/ ▪/ □/ ▪/ ▪/ □/ Genotypes^(c) ▪ □□ ▪ □ □ ▪ □ □ ▪ □ □ ▪ □ □ ▪ □ □ Taiwanese P₁ (n = 63) 5 58 0 5 58  0 558 0 4 59 0 4 59 0 4 59 0 P₂ (n = 164) 0 0 164 0 0 164 0 0 164 0 0 164 00 164 0 0 164 Indian P₁ (n = 20) 4 16 0 4 16  0 4 15 1 4 15 1 4 16 0 416 0 P₂ (n = 12) 0 0 12 0 0  12 0 0 12 0 0 12 0 0 12 0 0 12 Caucasian P₁(n = 32) 11 21 0 12 20  0 9 17 6 9 17 6 12 20 0 12 20 0 P₂ (n = 14) 0 014 0 0  14 0 0 14 0 0 14 0 0 14 0 0 14 African^(d) P₁ (n = 31) 11 17 313 17   1* 5 16 10 10 20 1 13 18 0 13 18 0 P₂ (n = 2) 0 0 2 0 0  2 0 0 20 0 2 0 0 2 0 0 2 Swedish^(e) P₁ (n = 151) 57 93  1 P₂ (n = 57) 0 0  57SNP7 SNP8 SNP9 SNP10 SNP11 Location^(a) +3443 +5012 +6762 +7038 +11507SNP ID rs9623671 rs111626860 rs5758891 rs8139674 rs66463955 Nucleotide GACTCCA T G G Polymorphisms^(c) (▪)/A(□) (▪)/—(□) (▪)/C(□) (▪)/T(□)(▪)/—(□) ▪/ ▪/ □/ ▪/ ▪/ □/ ▪/ ▪/ □/ ▪/ ▪/ □/ ▪/ ▪/ □/ Genotypes^(c) ▪ □□ ▪ □ □ ▪ □ □ ▪ □ □ ▪ □ □ Taiwanese P₁ (n = 63) 4 59 0 4 59 0 4 59 0 459 0 4 59 0 P₂ (n = 164) 0 0 164 0 0 164 0 1 163 0 5 159 0 2 162 IndianP₁ (n = 20) 4 15 1 4 15 1 4 15 1 6 13 1 4 15 1 P₂ (n = 12) 0 0 12 0 0 120 0 12 0 0 12 0 0 12 Caucasian P₁ (n = 32) 9 17 6 9 17 6 8 18 6 8 20 6 818 6 P₂ (n = 14) 0 0 14 0 0 14 0 0 14 0 2 10 0 1 13 African^(d) P₁ (n =31) 10 20 1 9 21 1 11 20 0 18 13 0 12 19 0 P₂ (n = 2) 0 0 2 0 0 2 0 0 20 0 2 0 0 2 Swedish^(e) P₁ (n = 151) P₂ (n = 57) The numbers of theindividuals that show genotype-phenotype inconsistency compared to themajority of others are typed in red. ^(a)Transcription initiationnucleotide of A4GALT exon 1 (74 bp) of RefSeq Transcript NM_017436.4 inthe NCBI database as +1 ^(b)SNPs rs67162484 and rs66781836 yieldidentical sequence polymorphisms. ^(c)The two different nucleotidegenotypes at each SNP are denoted by ▪ and □. ^(d)An asterisk indicatesa P₁ African who shows a genotype-phenotype discrepancy at SNP2.^(e)Ref. 28. The association dataset for the other SNPs in thispopulation is not available.

By a pilot investigation and stepwise SNP screening analysis in A4GALTgene followed by an association study using four ethnic populations, thepresent invention confirms that SNPs rs2143918 and rs5751348 arecorrelated with the P1 and P2 blood type. Accordingly, the T/T and T/Ggenotypes at the SNP rs2143918 are associated with P₁ phenotype whilethe G/G genotype at the SNP rs2143918 is associated with P₂ phenotype.The G/G and G/T genotypes at the SNP rs5751348 are associated with P₁phenotype while the T/T genotype at the SNP rs5751348 is associated withP₂ phenotype.

While some of the embodiments of the present invention have beendescribed in detail in the above, it is, however, possible for those ofordinary skill in the art to make various modifications and changes tothe particular embodiments shown without substantially departing fromthe teaching and advantages of the present invention. Such modificationsand changes are encompassed in the spirit and scope of the presentinvention as set forth in the appended claim.

What is claimed is:
 1. A method for determining P₁/P₂ blood type,comprising steps of: providing a biological sample of a subject;detecting a genotype for a single nucleotide polymorphism at rs2143918or rs5751348 in A4GALT gene of the biological sample; and determining aphenotype of the subject based on the genotype.
 2. The method of claim1, wherein the genotype of the single nucleotide polymorphism atrs2143918 is selected from the group consisting of T/T, T/G and GIG. 3.The method of claim 1, wherein T/T or T/G genotype of the singlenucleotide polymorphism at rs2143918 represents P₁ phenotype in thesubject.
 4. The method of claim 1, wherein G/G genotype of the singlenucleotide polymorphism at rs2143918 represents P₂ phenotype in thesubject.
 5. The method of claim 1, wherein the genotype of the singlenucleotide polymorphism at rs5751348 is selected from the groupconsisting of G/G, G/T and T/T.
 6. The method of claim 1, wherein G/G orG/T genotype of the single nucleotide polymorphism at rs5751348represents P₁ phenotype in the subject.
 7. The method of claim 1,wherein T/T genotype of the single nucleotide polymorphism at rs5751348represents P₂ phenotype in the subject.
 8. The method of claim 1,wherein the biological sample is blood or saliva.
 9. The method of claim1, wherein a nucleic acid in the biological sample is determined bypolymerase chain reaction (PCR).
 10. The method of claim 9, wherein aprimer pair of SEQ ID Nos: 1 and 2 are used in the PCR.
 11. The methodof claim 9, wherein a primer pair of SEQ ID Nos: 3 and 4 are used in thePCR.
 12. A detection kit for determining P₁/P₂ blood type, comprising: aprimer pair for determining a genotype of a single nucleotidepolymorphism at rs2143918 or rs5751348 of A4GALT gene in a nucleic acidsample of a subject.
 13. The detection kit of claim 12, wherein theprimer pair has sequences of SEQ ID Nos: 1 and
 2. 14. The detection kitof claim 12, wherein the primer pair has sequences of SEQ ID Nos: 3 and4.
 15. The detection kit of claim 12, wherein the genotype of the singlenucleotide polymorphism at rs2143918 is selected from the groupconsisting of T/T, T/G and G/G.
 16. The detection kit of claim 15,wherein T/T or T/G genotype of the single nucleotide polymorphism atrs2143918 represents P₁ phenotype in the subject.
 17. The detection kitof claim 15, wherein G/G genotype of the single nucleotide polymorphismat rs2143918 represents P₂ phenotype in the subject.
 18. The detectionkit of claim 12, wherein the genotype of the single nucleotidepolymorphism at rs5751348 is selected from the group consisting of G/G,G/T and T/T.
 19. The detection kit of claim 18, wherein G/G or G/Tgenotype of the single nucleotide polymorphism at rs5751348 representsP₁ phenotype in the subject.
 20. The detection kit of claim 18, whereinT/T genotype of the single nucleotide polymorphism at rs5751348represents P₂ phenotype in the subject.